Gestational jet lag predisposes to later-life skeletal and cardiac disease.

Circadian rhythm disturbance (CRD) increases the risk of disease, e.g. metabolic syndrome, cardiovascular disease, and cancer. In the present study, we investigated later life adverse health effects triggered by repeated jet lag during gestation. Pregnant mice were subjected to a regular light-dark cycle (CTRL) or to a repeated delay (DEL) or advance (ADV) jet lag protocol. Both DEL and ADV offspring showed reduced weight gain. ADV offspring had an increased circadian period, and an altered response to a jet lag was observed in both DEL and ADV offspring. Analysis of the bones of adult male ADV offspring revealed reduced cortical bone mass and strength. Strikingly, analysis of the heart identified structural abnormalities and impaired heart function. Finally, DNA methylation analysis revealed hypermethylation of miR17-92 cluster and differential methylation within circadian clock genes, which correlated with altered gene expression. We show that developmental CRD affects the circadian system and predisposes to non-communicable disease in adult life.

Mucin 1 (Muc1) Deficiency in Female Mice Leads to Temporal Skeletal Changes During Aging.

Mucin1 (MUC1) encodes a glycoprotein that has been demonstrated to have important roles in cell-cell interactions, cell-matrix interactions, cell signaling, modulating tumor progression and metastasis, and providing physical protection to cells against pathogens. In this study, we investigated the bone phenotype in female C57BL/6 Muc1 null mice and the impact of the loss of Muc1 on osteoblasts and osteoclasts. We found that deletion of Muc1 results in reduced trabecular bone volume in 8-week-old mice compared with wild-type controls, but the trabecular bone volume fraction normalizes with increasing age. In mature female mice (16 weeks old), Muc1 deletion results in stiffer femoral bones with fewer osteoblasts lining the trabecular surface but increased endosteal mineralized surface and bone formation rate. The latter remains higher compared with wild-type females at age 52 weeks. No difference was found in osteoclast numbers in vivo and in bone marrow osteoblast or osteoclast differentiation capacity or activity in vitro. Taken together, these results suggest that Muc1 depletion causes a transiently reduced trabecular bone mass phenotype in young mice, and later in life reduced numbers of osteoblasts with increased endocortical mineralization activity coincides with unaffected total bone mass and increased stiffness. In conclusion, our results show, for the first time to our knowledge, a role for Muc1 in bone mass and mineralization in mice in a time-dependent manner. © 2018 The Authors. JBMR Plus published by Wiley Periodicals, Inc. on behalf of American Society for Bone and Mineral Research.

Hydroxychloroquine affects bone resorption both in vitro and in vivo.

We recently showed that patients with primary Sjögren syndrome (pSS) have significantly higher bone mineral density (BMD) compared to healthy controls. The majority of those patients (69%) was using hydroxychloroquine (HCQ), which may have favorable effects on BMD. The aim of the study was to evaluate whether HCQ modulates osteoclast function. Osteoclasts were cultured from PBMC-sorted monocytes for 14 days and treated with different HCQ doses (controls 1 and 5 µg/ml). TRAP staining and resorption assays were performed to evaluate osteoclast differentiation and activity, respectively. Staining with an acidification marker (acridine orange) was performed to evaluate intracellular pH at multiple timepoints. Additionally, a fluorescent cholesterol uptake assay was performed to evaluate cholesterol trafficking. Serum bone resorption marker ß-CTx was evaluated in rheumatoid arthritis patients. HCQ inhibits the formation of multinuclear osteoclasts and leads to decreased bone resorption. Continuous HCQ treatment significantly decreases intracellular pH and significantly enhanced cholesterol uptake in mature osteoclasts along with increased expression of the lowdensity lipoprotein receptor. Serum ß-CTx was significantly decreased after 6 months of HCQ treatment. In agreement with our clinical data, we demonstrate that HCQ suppresses bone resorption in vitro and decreases the resorption marker ß-CTx in vivo. We also showed that HCQ decreases the intracellular pH in mature osteoclasts and stimulates cholesterol uptake, suggesting that HCQ induces osteoclastic lysosomal membrane permeabilization (LMP) leading to decreased resorption without changes in apoptosis. We hypothesize that skeletal health of patients with increased risk of osteoporosis and fractures may benefit from HCQ by preventing BMD loss.

Using the Connectivity Map to discover compounds influencing human osteoblast differentiation.

Osteoporosis is a common skeletal disorder characterized by low bone mass leading to increased bone fragility and fracture susceptibility. Identification of factors influencing osteoblast differentiation and bone formation is very important. Previously, we identified parbendazole to be a novel compound that stimulates osteogenic differentiation of human mesenchymal stromal cells (hMSCs), using gene expression profiling and bioinformatic analyzes, including the Connectivity Map (CMap), as an in-silico approach. The aim for this paper is to identify additional compounds affecting osteoblast differentiation using the CMap. Gene expression profiling was performed on hMSCs differentiated to osteoblasts using Illumina microarrays. Our osteoblast gene signature, the top regulated genes 6 hr after induction by dexamethasone, was uploaded into CMap (www.broadinstitute.org/cmap/). Through this approach we identified compounds with gene signatures positively correlating (withaferin-A, calcium folinate, amylocaine) or negatively correlating (salbutamol, metaraminol, diprophylline) to our osteoblast gene signature. All positively correlating compounds stimulated osteogenic differentiation, as indicated by increased mineralization compared to control treated cells. One of three negatively correlating compounds, salbutamol, inhibited dexamethasone-induced osteoblastic differentiation, while the other two had no effect. Based on gene expression data of withaferin-A and salbutamol, we identified HMOX1 and STC1 as being strongly differentially expressed . shRNA knockdown of HMOX1 or STC1 in hMSCs inhibited osteoblast differentiation. These results confirm that the CMap is a powerful approach to identify positively compounds that stimulate osteogenesis of hMSCs, and through this approach we can identify genes that play an important role in osteoblast differentiation and could be targets for novel bone anabolic therapies.

Identification of Chloride Intracellular Channel Protein 3 as a Novel Gene Affecting Human Bone Formation.

Osteoporosis is a common skeletal disorder characterized by low bone mass leading to increased bone fragility and fracture susceptibility. The bone building cells, osteoblasts, are derived from mesenchymal stromal cells (MSCs); however, with increasing age osteogenic differentiation is diminished and more adipocytes are seen in the bone marrow, suggesting a shift in MSC lineage commitment. Identification of specific factors that stimulate osteoblast differentiation from human MSCs may deliver therapeutic targets to treat osteoporosis. The aim of this study was to identify novel genes involved in osteoblast differentiation of human bone marrow-derived MSCs (hMSCs). We identified the gene chloride intracellular channel protein 3 (CLIC3) to be strongly upregulated during MSC-derived osteoblast differentiation. Lentiviral overexpression of CLIC3 in hMSCs caused a 60% increase of matrix mineralization. Conversely, knockdown of CLIC3 in hMSCs using two short-hairpin RNAs (shRNAs) against CLIC3 resulted in a 69% to 76% reduction in CLIC3 mRNA expression, 53% to 37% less alkaline phosphatase (ALP) activity, and 78% to 88% less matrix mineralization compared to scrambled control. Next, we used an in vivo human bone formation model in which hMSCs lentivirally transduced with the CLIC3 overexpression construct were loaded onto a scaffold (hydroxyapatite-tricalcium-phosphate), implanted under the skin of NOD-SCID mice, and analyzed for bone formation 8 weeks later. CLIC3 overexpression led to a 15-fold increase in bone formation (0.33% versus 5.05% bone area relative to scaffold). Using a Clic3-His-tagged pull-down assay and liquid chromatography-mass spectrometry (LS/MS)-based proteomics analysis in lysates of osteogenically differentiated hMSCs, we showed that CLIC3 interacts with NIMA-related kinase 9 (NEK9) and phosphatidylserine synthase 1 (PTDSS1) in vitro, and this finding was supported by immunofluorescent analysis. In addition, inhibition of NEK9 or PTDSS1 gene expression by shRNAs inhibited osteoblast differentiation and mineralization. In conclusion, we successfully identified CLIC3 to be a lineage-specific gene regulating osteoblast differentiation and bone formation through its interaction with NEK9 and PTDSS1. © The Authors. JBMR Plus is published by Wiley Periodicals, Inc. on behalf of the American Society for Bone and Mineral Research.

Lifelong challenge of calcium homeostasis in male mice lacking TRPV5 leads to changes in bone and calcium metabolism.

Trpv5 plays an important role in calcium (Ca2+) homeostasis, among others by mediating renal calcium reabsorption. Accordingly, Trpv5 deficiency strongly stresses Ca2+ homeostasis in order to maintain stable serum Ca2+. We addressed the impact of lifelong challenge of calcium homeostasis on the bone phenotype of these mice.Aging significantly increased serum 1,25(OH)2D3 and PTH levels in both genotypes but they were more elevated in Trpv5-/- mice, whereas serum Ca2+ was not affected by age or genotype. Age-related changes in trabecular and cortical bone mass were accelerated in Trpv5-/- mice, including reduced trabecular and cortical bone thickness as well as reduced bone mineralization. No effect of Trpv5 deficiency on bone strength was observed. In 78-week-old mice no differences were observed between the genotypes regarding urinary deoxypyridinoline, osteoclast number, differentiation and activity as well as osteoclast precursor numbers, as assessed by flow cytometry.In conclusion, life-long challenge of Ca2+ homeostasis present in Trpv5-/- mice causes accelerated bone aging and a low cortical and trabecular bone mass phenotype. The phenotype of the Trpv5-/- mice suggests that maintenance of adequate circulatory Ca2+ levels in patients with disturbances in Ca2+ homeostasis should be a priority in order to prevent bone loss at older age.

Adverse Effects of Diabetes Mellitus on the Skeleton of Aging Mice.

In the present study, the possibility that a diabetic (DM) status might worsen age-related bone deterioration was explored in mice. Male CD-1 mice aged 2 (young control group) or 16 months, nondiabetic or made diabetic by streptozotocin injections, were used. DM induced a decrease in bone volume, trabecular number, and eroded surface, and in mineral apposition and bone formation rates, but an increased trabecular separation, in L1-L3 vertebrae of aged mice. Three-point bending and reference point indentation tests showed slight changes pointing to increased frailty and brittleness in the mouse tibia of diabetic old mice. DM was related to a decreased expression of both vascular endothelial growth factor and its receptor 2, which paralleled that of femoral vasculature, and increased expression of the pro-adipogenic gene peroxisome proliferator-activated receptor γ and adipocyte number, without affecting ß-catenin pathway in old mouse bone. Concomitant DM in old mice failed to affect total glutathione levels or activity of main anti-oxidative stress enzymes, although xanthine oxidase was slightly increased, in the bone marrow, but increased the senescence marker caveolin-1 gene. In conclusion, DM worsens bone alterations of aged mice, related to decreased bone turnover and bone vasculature and increased senescence, independently of the anti-oxidative stress machinery.

Connectivity Map-based discovery of parbendazole reveals targetable human osteogenic pathway.

Osteoporosis is a common skeletal disorder characterized by low bone mass leading to increased bone fragility and fracture susceptibility. In this study, we have identified pathways that stimulate differentiation of bone forming osteoblasts from human mesenchymal stromal cells (hMSCs). Gene expression profiling was performed in hMSCs differentiated toward osteoblasts (at 6 h). Significantly regulated genes were analyzed in silico, and the Connectivity Map (CMap) was used to identify candidate bone stimulatory compounds. The signature of parbendazole matches the expression changes observed for osteogenic hMSCs. Parbendazole stimulates osteoblast differentiation as indicated by increased alkaline phosphatase activity, mineralization, and up-regulation of bone marker genes (alkaline phosphatase/ALPL, osteopontin/SPP1, and bone sialoprotein II/IBSP) in a subset of the hMSC population resistant to the apoptotic effects of parbendazole. These osteogenic effects are independent of glucocorticoids because parbendazole does not up-regulate glucocorticoid receptor (GR) target genes and is not inhibited by the GR antagonist mifepristone. Parbendazole causes profound cytoskeletal changes including degradation of microtubules and increased focal adhesions. Stabilization of microtubules by pretreatment with Taxol inhibits osteoblast differentiation. Parbendazole up-regulates bone morphogenetic protein 2 (BMP-2) gene expression and activity. Cotreatment with the BMP-2 antagonist DMH1 limits, but does not block, parbendazole-induced mineralization. Using the CMap we have identified a previously unidentified lineage-specific, bone anabolic compound, parbendazole, which induces osteogenic differentiation through a combination of cytoskeletal changes and increased BMP-2 activity.

Identification of microRNAs in human plasma.

In recent years, microRNAs (miRNA) have been demonstrated to be present in body fluids and may therefore serve as diagnostic markers for diseases. By characterizing miRNA profiles in plasma, a miRNA signature may potentially be developed as a diagnostic and risk assessment tool for particular (patho)physiological states. This chapter describes the isolation, purification, identification, and sequencing of human plasma miRNAs.

A human vitamin D receptor mutation causes rickets and impaired Th1/Th17 responses.

We present a brother and sister with severe rickets, alopecia and highly elevated serum levels of 1,25-dihydroxyvitamin D (1,25-(OH)2D3). Genomic sequencing showed a homozygous point mutation (A133G) in the vitamin D receptor gene, leading to an amino acid change in the DNA binding domain (K45E), which was described previously. Hereditary vitamin D resistant rickets (HVDRR) was diagnosed. Functional studies in skin biopsy fibroblasts confirmed this. 1,25-(OH)2D3 reduced T helper (Th) cell population-specific cytokine expression of interferon γ (Th1), interleukins IL-17A (Th17) and IL-22 (Th17/Th22) in peripheral blood mononuclear cells (PBMCs) from the patient's parents, whereas IL-4 (Th2) levels were higher, reflecting an immunosuppressive condition. None of these factors were regulated by 1,25-(OH)2D3 in PBMCs from the boy. At present, both patients (boy is 23 years of age, girl is 7) have not experienced any major immune-related disorders. Although both children developed alopecia, the girl did so earlier than the boy. The boy showed complete recovery from the rickets at the age of 17 and does not require any vitamin D supplementations to date. In conclusion, we characterized two siblings with HVDRR, due to a mutation in the DNA binding domain of VDR. Despite a defective T cell response to vitamin D, no signs of any inflammatory-related abnormalities were seen, thus questioning an essential role of vitamin D in the immune system. Despite the fact that currently medicine is not required, close monitoring in the future of these patients is warranted for potential recurrence of vitamin D dependence and diagnosis of (chronic) inflammatory-related diseases.

The transient receptor potential channel TRPV6 is dynamically expressed in bone cells but is not crucial for bone mineralization in mice.

Bone is the major store for Ca(2+) in the body and plays an important role in Ca(2+) homeostasis. During bone formation and resorption Ca(2+) must be transported to and from bone by osteoblasts and osteoclasts, respectively. However, little is known about the Ca(2+) transport machinery in these bone cells. In this study, we examined the epithelial Ca(2+) channel TRPV6 in bone. TRPV6 mRNA is expressed in human and mouse osteoblast-like cells as well as in peripheral blood mononuclear cell-derived human osteoclasts and murine tibial bone marrow-derived osteoclasts. Also other transcellular Ca(2+) transport genes, calbindin-D(9k) and/or -D(28K), Na(+)/Ca(2+) exchanger 1, and plasma membrane Ca(2+) ATPase (PMCA1b) were expressed in these bone cell types. Immunofluorescence and confocal microscopy on human osteoblasts and osteoclasts and mouse osteoclasts revealed TRPV6 protein at the apical domain and PMCA1b at the osteoidal domain of osteoblasts, whereas in osteoclasts TRPV6 was predominantly found at the bone-facing site. TRPV6 was dynamically expressed in human osteoblasts, showing maximal expression during mineralization of the extracellular matrix. 1,25-Dihydroxyvitamin D(3) (1,25(OH)(2)D(3)) did not change TRPV6 expression in both mineralizing and non-mineralizing SV-HFO cultures. Lentiviral transduction-mediated overexpression of TRPV6 in these cells did not alter mineralization. Bone microarchitecture and mineralization were unaffected in Trpv6(D541A/D541A) mice in which aspartate 541 in the pore region was replaced with alanine to render TRPV6 channels non-functional. In summary, TRPV6 and other proteins involved in transcellular Ca(2+) transport are dynamically expressed in bone cells, while TRPV6 appears not crucial for bone metabolism and matrix mineralization in mice.